The present application claims priority to co-pending German Patent Application No. 19920611.2, filed May 5, 1999, which is incorporated by reference herein in its entirety.
The invention originates from the field of cloning and amplification of nucleic acids and concerns a method for cloning cDNAs that are complete at the 5xe2x80x2 end.
The molecular analysis of messenger RNAs (mRNAs) that have been transcribed in vivo is usually carried out by generating and cloning so-called cDNAs. For this a previously isolated, poly-adenylated mRNA is firstly reversely transcribed using an oligo-dT primer i.e. it is transcribed into a single-stranded cDNA which is complementary to the mRNA. Subsequently a second strand which is complementary to the single-stranded cDNA is polymerized by methods known to a person skilled in the art to form a double-stranded cDNA. After amplifying and cloning the cDNA or parts of the cDNA using conventional molecular-biological methods (cf. Sambrook, Molecular Cloning, Laboratory Manual, 2nd edition, chapter 14), it is possible to subsequently determine the sequence of the mRNA. However, a disadvantage of this method is that it is seldom possible to identify and clone cDNAs which have a complete 5xe2x80x2 end or can only be achieved with a very low efficiency. The reason for this is an inefficient reverse transcription reaction due to formation of intramolecular secondary structures as well as a limited amount of starting material whose RNA content is of inadequate quality. An additional disadvantage is that a loss of terminal 5xe2x80x2 sequences of the mRNA occurs in conventional methods due to the manner in which the second strand is synthesized.
Various methods which have been developed in the past to overcome this problem have concentrated on using the cap structure of the mRNA which occurs at the 5xe2x80x2 end of complete cDNAs as an additional selection criterion. This is a terminal guanosine residue which is methylated at position 7 and is linked by a 5xe2x80x2xe2x80x945xe2x80x2 bond to the actual mRNA.
In the oligo capping method (Maruyama and Sugaru, Gene 138, 171-174, 1994) the cap structure is firstly removed enzymatically by a suitable phosphatase treatment and replaced by an oligonucleotide which is linked to the 5xe2x80x2 end of the mRNA by a suitable ligation step. However, this method requires many enzymatic steps and a large amount of poly-A-mRNA as the starting material.
Another method, the Cap Finder method (Clontech, Clontechniques 11, 1 (1996), Maleszka and Stange, Gene 202, 39-43 (1997)) is based on a marginal terminal transferase activity of the reverse transcriptase which leads to a so-called template switching effect: In a first strand cDNA synthesis a few deoxy-cytosine residues are added selectively to the 3xe2x80x2 end of the cDNA by the reverse transcriptase under suitable conditions. This produces an anchor sequence for a so-called template switching oligonucleotide with a 3xe2x80x2 end composed of guanosine residues which, after hybridization to the anchor sequence, serves as a primer for the second strand synthesis of the cDNA. However, the terminal transferase activity of the reverse transcriptase which apparently begins at the 7-methyl-guanosine-cap structure of the mRNA and adds the cytosine residues, has previously not been characterized in detail so that the influence of certain secondary structures of the mRNA template or of the cDNA end on the template switching activity was unknown at the time of the invention and according to the prior art the reaction could only be carried out with a low efficiency.
In an alternative method (WO 97/26368) a suitable anchor sequence is synthesized with the aid of a terminal transferase enzyme which is different from the reverse transcriptase in the process of which two to four ribonucleotides (instead of deoxyribonucleotides) are attached as an anchor sequence to the 3xe2x80x2 end of the first strand cDNA. However, a disadvantage of this method is that the cDNA synthesis cannot be carried out selectively for 5xe2x80x2-cap mRNAs.
Hence the technical object of the invention was to develop an additional method for the modification, cloning or amplification of cDNAs which is used to obtain cDNAs that have a complete 5xe2x80x2 end in the simplest possible manner and as efficiently as possible.
This technical object is achieved in that the terminal transferase reaction that elongates the first strand cDNA with deoxy-cytosines is carried out by a reverse transcriptase in the presence of magnesium2+ ions as well as in the presence of manganese2+ ions. The manganese concentration to be used is preferably 1-20 mM and under optimized conditions 8 mM.
In one embodiment the manganese2+ ions can already be contained in the buffer system used during the cDNA first strand synthesis. Alternatively an incubation in the presence of manganese2+ ions can be carried out directly after a first strand cDNA synthesis carried out in the absence of manganese2+ ions in which case the reverse transcriptase used originally is still active under these conditions.
In this process the reverse transcriptase develops a terminal transferase activity which leads to an efficient addition of 2 to 4 deoxy-cytosine residues at the 3xe2x80x2 terminus of the newly synthesized cDNA. This reaction is extremely efficient and also dependent on the presence of the cap structure at the 5xe2x80x2 end of the mRNA template.
In both embodiments the deoxy-nucleotide tailing in the presence of Mn2+ ions according to the invention not only results in a high efficiency of the tailing reaction. At the same time the specificity of the reaction is retained i.e. the addition of two to four deoxy-cytosine residues in the presence of a 5xe2x80x2-cap structure on the mRNA template.
Any reverse transcriptase enzymes can be used with the only restriction that the enzyme that is used should not have any RNAseH activity.
A so-called xe2x80x9canchor primerxe2x80x9d is preferably used as the primer for the first strand synthesis which is composed of two parts: a so-called xe2x80x9canchor sequencexe2x80x9d is located at the 5xe2x80x2 end which can serve as a target sequence for PCR primers in amplification reactions that take place at a later time. In contrast the 3xe2x80x2 terminal end is composed of a sequence that can hybridize with the mRNA to be identified. This can for example be an oligo-dT sequence which hybridizes with the poly-A tail 3xe2x80x2 end of the mRNA.
Following a cDNA synthesis according to the invention, the first strand cDNA is reacted in a preferred embodiment with a ribonucleotide triphosphate or comparable derivatives such as 2xe2x80x2-O-methyl or 2xe2x80x2-O-amino nucleotide triphosphates in the presence of a terminal transferase that is different from reverse transcriptase to form an anchor sequence of ribonucleotide residues at its 3xe2x80x2 end. When a certain ribonucleotide triphosphate is used, the reaction is referred to as controlled ribonucleotide tailing (CTRT). The ribonucleotide is preferably not CTP; the use of ATP is particularly advantageous.
The present invention also concerns methods in which the product of the reaction is subsequently linked to an additional double-stranded nucleic acid molecule which has a 3xe2x80x2-overhanging end that is complementary to the 3xe2x80x2 end of the product of the reaction. Suitable additional double-stranded nucleic acid molecules are for example DNA vectors or short adaptor molecules which have target sequences for PCR primers for the subsequent amplification.
Furthermore the double-stranded nucleic acid molecules that are used can contain sequences which facilitate a subsequent analysis after the amplification is completed. These for example include promoter sequences suitable for in vitro transcription such as the prokaryotic T7, T3 or SP6 promoters and also restriction cleavage sites of which rare cleavage sites for so-called rare cutter enzymes such as NotI are particularly preferred.
The additional double-stranded nucleic acid molecule is preferably ligated to the 3xe2x80x2 end of the product of the above mentioned reaction. In this connection it has turned out that hybridization of the adaptor to the first strand cDNA is particularly efficient in the presence of standard ligation buffers containing DMSO and efficiencies of more than 90% can be achieved. Concentrations of 5-10 vol. % DMSO have proven to be advantageous. A concentration of 7.5 vol % was determined during the optimization of the reaction.
This step includes the actual selection of full-length cDNA molecules which have the specific 3xe2x80x2 overhangs as a result of the selection of adaptors or vectors. These overhangs are essentially characterized in that they are completely complementary to the 3xe2x80x2 end of the initially synthesized full-length cDNA. This is accomplished by the overhang being composed of 3 or 4 deoxy-thymidine residues followed by 3 deoxy-guanosine residues when ATP is used for the terminal transferase reaction.
Due to the fact that the number of nucleotide residues added to each cDNA molecule during the tailing reaction of the reverse transcriptase and during the controlled ribonucleotide tailing is not completely identical, it has proven to be advantageous when the adaptors are composed of a mixture of molecules whose 3xe2x80x2 overhangs vary between 3 and 4 deoxy-thymidine residues and possibly also between 3 and 4 deoxy-guanosine residues.
Consequently a mixture of double-stranded DNA adaptors containing 3xe2x80x2 overhangs composed of 5xe2x80x2-dT3-4dG3-3xe2x80x2 is also a subject matter of the invention. The invention also concerns single-stranded cDNAs which have a non-mRNA template coded 3xe2x80x2 end composed of 5xe2x80x2-dC3-4-rA3-4-3xe2x80x2.
In the subsequent amplification according to the invention using PCR it is possible to distinguish between several basic methods:
A) Construction of a cDNA Gene Bank
A primer pair comprising an adaptor primer and a so-called anchor primer is used for this. As described above the adaptor primer that is used is directed against the double-stranded sequence of the DNA adaptor molecule. In contrast the sequence of the anchor primer corresponds to the anchor part that was attached to the 5xe2x80x2 end of the cDNA during the first strand cDNA synthesis by the reverse transcriptase reaction. The pool of amplification products formed in this manner potentially contains numerous different full-length cDNAs and can be used to generate a cDNA gene bank by conventional molecular biological methods.
FIG. 1 shows a schematic overview of the method according to the invention: The method combines the 5xe2x80x2 CAP-dependent addition of 3-4 cytosine residues to the 3xe2x80x2 terminus of full-length first strand cDNAs by the reverse transcriptase (RT) (1., with the inventive use of a reaction buffer containing manganese chloride) with the technique of controlled ribonucleotide tailing of cDNA ends (CRTC) by the enzyme terminal transferase and rATP (2.). The terminal sequence motif (5xe2x80x2-dC3rA3-4) formed in this manner enables the full-length cDNA to be selectively ligated to a double-stranded DNA adaptor (5xe2x80x2-dT3-4dG3) using T4 DNA ligase (3.). This method leads to the specific amplification of full-length cDNAs which contain a complete 5xe2x80x2 sequence of the mRNA. Subsequently a cDNA bank can be constructed (5.2.) after a suitable amplification by PCR.
B) Isolation and Amplification of a Specific mRNA for which Partial Sequence Information is Available.
In this step a so-called adaptor primer and an mRNA primer are used as the primers for amplification. The adaptor primer is directed against the double-stranded region of the DNA adaptor molecule. In contrast the mRNA primer is complementary to a previously known sequence section of the mRNA. The amplification products obtained in this manner represent cDNAs which are complete at their 5xe2x80x2 end but have a sequence section of a particular length missing at their 3xe2x80x2 end that depends on the choice of the mRNA primer.
If the mRNA to be identified is a weakly expressed species, it can be amplified again in a nested PCR using an additional primer pair of which one primer is in turn directed against the double-stranded adaptor sequence and an additional primer is directed against an internal sequence of the mRNA.
In a special embodiment the adaptor primer can contain an immobilizable label such as biotin so that the amplification products that are formed can be bound to a solid phase coated with streptavidin. In this manner the amplification products can for example be more simply analysed by direct sequencing.
The invention additionally concerns methods in which the cDNA is amplified using a primer provided with an immobilizable group. In an alternative embodiment the double-stranded DNA adaptor molecule is, by contrast, provided with an immobilizable group. As a result the amplification product, whose generation is dependent on cDNA, can be coupled to a solid phase for further analysis.
The invention additionally concerns in particular methods which are characterized in that the amplification reaction is carried out several times. This repeated amplification can be carried out with several amplification primers in the form of a nested PCR.
The invention also concerns particular embodiments of the inventive method in which the amplification product is subsequently directly sequenced.
In addition to the said methods, other amplification methods are also a subject matter of the invention which are based on special analytical formats such as differential display (WO 93/18176), serial analysis of gene expression (U.S. Pat. No. 5,695,937), subtractive hybridization (Wang et al., Proc. Natl. Acad. Sci. USA 88:11505 (1991)) or linear amplification with the aid of T7 RNA polymerase.